The Bilateral Ovariectomy in a Female Animal Exacerbates the Pathogenesis of an Intracranial Aneurysm - MDPI
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brain
sciences
Article
The Bilateral Ovariectomy in a Female Animal
Exacerbates the Pathogenesis of an
Intracranial Aneurysm
Mieko Oka 1,2,3 , Isao Ono 1,2,4 , Kampei Shimizu 1,2,4 , Mika Kushamae 1,2,5 ,
Haruka Miyata 1,2,6 , Takakazu Kawamata 3 and Tomohiro Aoki 1,2, *
1 Department of Molecular Pharmacology, Research Institute, National Cerebral and Cardiovascular Center,
Osaka 564-8565, Japan; happy.harmony.toto@gmail.com (M.O.); onoisao@kuhp.kyoto-u.ac.jp (I.O.);
k.shimizu.830923@gmail.com (K.S.); marojiji9@yahoo.co.jp (M.K.); hmiyata27@gmail.com (H.M.)
2 Core Research for Evolutional Science and Technology (CREST) from Japan Agency for Medical Research
and Development (AMED), National Cerebral and Cardiovascular Center, Osaka 564-8565, Japan
3 Department of Neurosurgery, Tokyo Women’s Medical University, Tokyo 162-8666, Japan;
tkawamata@twmu.ac.jp
4 Department of Neurosurgery, Kyoto University Graduate School of Medicine, Tokyo 606-8507, Japan
5 Department of Neurosurgery, Showa University, Tokyo 142-8666, Japan
6 Department of Neurosurgery, Shiga University of Medical Science, Shiga 520-2192, Japan
* Correspondence: tomoaoki@ncvc.go.jp; Tel.: +81-6-6170-1070 (ext. 31022)
Received: 24 April 2020; Accepted: 28 May 2020; Published: 31 May 2020
Abstract: Considering the poor outcome of subarachnoid hemorrhage (SAH) due to the rupture
of intracranial aneurysms (IA), mechanisms underlying the pathogenesis of IAs, especially the
rupture of lesions, should be clarified. In the present study, a rat model of IAs in which induced
lesions spontaneously ruptured resulting in SAH was used. In this model, the combination of the
female sex and the bilateral ovariectomy increased the incidence of SAH, similar to epidemiological
evidence in human cases. Importantly, unruptured IA lesions induced in female animals with
bilateral ovariectomy were histopathologically similar to ruptured ones in the presence of vasa
vasorum and the accumulation of abundant inflammatory cells, suggesting the exacerbation of the
disease. The post-stenotic dilatation of the carotid artery was disturbed by the bilateral ovariectomy in
female rats, which was restored by hormone replacement therapy. The in vivo study thus suggested
the protective effect of estrogen from the ovary on endothelial cells loaded by wall shear stress.
β-estradiol or dihydrotestosterone also suppressed the lipopolysaccharide-induced expression of
pro-inflammatory genes in cultured macrophages and neutrophils. The results of the present study
have thus provided new insights about the process regulating the progression of the disease.
Keywords: intracranial aneurysm; subarachnoid hemorrhage; estrogen; female; endothelial
cell; macrophage
1. Introduction
Considering the devastating outcome of subarachnoid hemorrhage (SAH) due to the rupture
of an intracranial aneurysm (IA) [1,2], the development of a novel therapeutic strategy to prevent
the rupture of IAs is mandatory for social health. Mechanisms underlying the rupture of lesions
should, therefore, be clarified. Thus, we attempted to find a cue from well-established epidemiological
evidence. Epidemiological studies have consistently demonstrated a higher incidence of SAH in older
females or postmenopausal females [3–8]. Based on the necessity of clarifying underlying mechanisms
Brain Sci. 2020, 10, 335; doi:10.3390/brainsci10060335 www.mdpi.com/journal/brainsci
Brain Sci. 2020, 10, 335 2 of 11
of the rupture of IAs, we examined whether sex difference is indeed present, and if present, why, using
the already-established animal model of IAs [9].
2. Materials and Methods
2.1. IA Models of Rats and Histological Analysis of Induced IA
All of the following experiments, including animal care and use, complied with the National
Institute of Health’s Guide for the Care and Use of Laboratory Animals and complied with the
National Institute of Health’s Guide for the Care and Use of Laboratory Animals and were approved
by the Institutional Animal Care and Use Committee of the National Cerebral and Cardiovascular
Center (Approved number; #18010 and #19036). The present manuscript also adheres to the ARRIVE
(Animal Research: Reporting of In Vivo Experiments) guidelines for reporting animal experiments.
Ten-week-old male or female Sprague−Dawley (SD) rats were purchased from Japan SLC (Slc:SD,
Shizuoka, Japan) (n = 62 in total). Animals were maintained on a 12-h light/dark cycle, and had free
access to feed and water. To induce IAs, the rats were subjected to ligation of the left carotid artery,
the right external carotid artery and the right pterygopalatine artery, and systemic hypertension by
the combination of a high salt diet and the ligation of the left renal artery under general anesthesia
by the intraperitoneal injection of pentobarbital sodium (50 mg/kg, Somnopentyl, Kyoritsuseiyaku
Corporation, Tokyo, Japan) and the inhalation of Isoflurane (1.5%–2%, #IYESC-0001, Pfizer Inc.,
New York, NY). In some female rats, the bilateral ovariectomy was also applied [9]. Immediately
after the above surgical manipulations, animals were fed chow containing 8% sodium chloride and
0.12% 3-aminopropionitrile (#A0408, Tokyo Chemical Industry, Tokyo, Japan), an irreversible inhibitor
of lysyl oxidase catalyzing the cross-linking of collagen and elastin. Animals that died within one
week after the above surgical manipulations were excluded from the analyses. At 16 weeks after the
surgical manipulations, blood pressure was measured by a tail-cuff method without any anesthesia
and was calculated as an average of three measurements. Animals were then deeply anesthetized
by an intraperitoneal injection of pentobarbital sodium (200 mg/kg), and transcardially perfused
with 4% paraformaldehyde solution. The circle of Willis was then stripped from the brain surface
and an IA lesion induced at the anterior communicating artery or the posterior communicating
artery was dissected as a ruptured or unruptured lesion, according to the macroscopic observation
of whether the clot or hemosiderin deposition was present around IA lesions. Here, ruptured
IAs were exclusively induced at these sites examined in the model [9]. All the dead animals after
at least one week of surgical manipulations were autopsied to examine the onset of SAH due to
rupture of induced IAs. Histopathological examination was done after Elastica van Gieson, which
visualizes the internal elastic lamina using 5-um-thick frozen sections. The size of induced lesions was
analyzed using the slices with the maximum area selected from serial sections using ImageJ software
(https://imagej.nih.gov/ij/index.html).
2.2. Immunohistochemistry
At the indicated period after the aneurysm induction, 5-µm-thick frozen sections were prepared.
After blocking with 3% donkey serum (#AB_2337258, Jackson ImmunoResearch, Baltimore, MD, USA),
slices were incubated with primary antibodies, followed by incubation with secondary antibodies
conjugated with a fluorescence dye (Jackson ImmunoResearch). Finally, fluorescent images were
acquired using a confocal fluorescence microscope system (FV1000 or FV3000, Olympus, Tokyo, Japan).
The following primary antibodies were used: mouse monoclonal anti-CD68 antibody (#ab31630,
Abcam, Cambridge, UK), rabbit polyclonal anti-myeloperoxidase (MPO) antibody (#ab9535, Abcam),
rabbit polyclonal anti-tumor necrosis factor (TNF)-alpha antibody (#ab6671, Abcam), mouse monoclonal
anti-smooth muscle α-actin (SMA) antibody (#M0851, Dako, Agilent, Santa Clara, CA, USA).
The following secondary antibodies were used; Alexa Fluor 488-conjugated donkey anti-mouse
IgG H&L antibody (#A21202, Thermo Fisher Scientific, Waltham, MA, USA), Alexa Fluor 488-conjugatedBrain Sci. 2020, 10, 335 3 of 11
donkey anti-rabbit IgG H&L antibody (#A21206, Thermo Fisher Scientific), Alexa Fluor 594-conjugated
donkey anti-mouse IgG H&L antibody (#A21203, Thermo Fisher Scientific).
2.3. Stenosis Model of the Carotid Artery of a Rat
Female rats underwent a bilateral ovariectomy and sham operation, and were then maintained for
7 days before subjecting to the model. The left common carotid artery of rats was then ligated using a
10-0 nylon thread with 25 gauge needle put on the side of the artery and stenosis was established by
removing only the needle [10,11]. The post-stenotic dilatation of the carotid artery was observed for
30 min after ligation.
2.4. Hormone Replacement Therapy
Estradiol valerate (1 mg/kg, #224136400 Pelanin Depot, Mochida Pharmaceutical Co., Ltd.,
Tokyo, Japan) was intramuscularly injected every 7 days in a female rat that underwent the
bilateral ovariectomy.
2.5. Cell Line and Culture
RAW264.7 cell line (#TIB-71), used as a macrophage, and HL-60 cell line (#CCL-240), used as a
neutrophil, were purchased from ATCC (Manassas, VA, USA) and maintained in Dulbecco’s Modified
Eagle’s Medium (DMEM) (#044-32955, FUJIFILM Wako Pure Chemical Corporation, Osaka, Japan)
supplemented with 10% or 20% fetal bovine serum (#FB-1365/500, Biosera, Nuaille, France), respectively.
2.6. Quantitative Real Time (RT)-PCR Analysis in Cultured Cells
RAW264.7 cells or HL-60 cells were pre-treated with β-estradiol (E2, 50 µg/mL, #E0025, Tokyo
Chemical Industry) or 5α-Dihydrotestosterone (DHT, 50 µg/mL, #A0462, Tokyo Chemical Industry)
for 24 h or 3 h, respectively. Cells were then stimulated with vehicle (Veh), LPS (1 µg/mL, #L2654,
Sigma Aldrich, St. Louis, MO, USA) or TNF-α (100 ng/mL, R&D SYSTEMS, Minneapolis, MN, USA)
for additional 60 min.
Total RNA was purified from stimulated cells and reverse-transcribed using a RNeasy Mini Kit
(#74106, QIAGEN, Hilden, Germany) and a High-capacity cDNA Reverse Transcription Kit (#4368813,
Life Technologies Corporation, Carlsbad, CA, USA), according to the manufacturers’ instructions. For
quantification of gene expression, quantitative RT-PCR was performed on a LightCycler 480 (Roche,
Indianapolis, IN, USA) with a TB Green Premix Ex Taq II (#RR820, TAKARA BIO INC., Shiga, Japan).
Expression of Actb (a gene coding β-actin) in experiments using the RAW264.7 cell line, or ACTB in
experiments using the HL-60 cell line, were used as internal controls. For quantitation, the second
derivative maximum method was used for determining the crossing point.
Primer sets used are listed as follows: forward 50 -CACCTCAGGGAAGAATCTGG-30 and reverse
50 -CATTCCTGAGTTCTGCAAAGG-30 for Tnf ; forward 50 -AAAGGGAGCTCCTTAACATGC-30
and reverse 50 -CTTCCTGGGAAACAACAGTGG-30 for Il1b; forward
50 -AATGATGTGTACGGCTTCAGG-30 and reverse 50 -CTGTACAAGCAGTGGCAAAGG-30 for Ptgs2
(a gene encoding cyclooxygenase-2 (COX-2)); forward 50 -GCACAGACCTCTCTCTTGAGC-30
and reverse 50 -ACCTGCTGCTGCTACTCATTCACC-30 for Ccl2 (a gene encoding
monocyte chemoattractant protein-1 (MCP-1)); 50 -ACGACCAGAGGCATACAGGGA-30 and
50 -CCCTAAGGCCAACCGTGAAA-30 for Actb; forward 50 -TCAGCAATGAGTGACAGTTGG-30 and
reverse 50 -ATAGGCTGTTCCCATGTAGCC-30 for TNF; forward 50 -CAAGCTGGAATTTGAGTCTGC-30
and reverse 50 -ATTCAGCACAGGACTCTCTGG-30 for IL1B; forward
5 -ACACCCTCTATCACTGGCATCC-30 and reverse 50 -AACATTCCTACCACCAGCAACC-30 for
0
PTGS2; forward 50 -AGCTTCTTTGGGACACTTGC-30 and reverse 50 -ATAGCAGCCACCTTCATTCC-30
for CCL2 and forward 50 -CATACTCCTGCTTGCTGATCC-30 and reverse
0
5 -GATGCAGAAGGAGATCACTGC-3 for ACTB. 0Brain Sci. 2020, 10, 335 4 of 11
2.7. Statistical Analysis
Data are shown as the mean ± SEM. Statistical comparisons between two or more groups
were conducted using a Welch’s t-test or the Tukey-Kramer method, respectively, with JMP Pro 14
(SAS Institute Inc., Cary, NC, USA). A p value less than 0.05 was defined as statistically significant.
3. Results
3.1. Highest Incidence of SAH in Female Rats with the Bilateral Ovariectomy
In reference to established epidemiological evidence, the risk of SAH is higher in postmenopausal
or older females than in males and in females before menopause [3–8], we first examined whether the
sex difference in the incidence of SAH could be reproduced or not in a rat model of IAs [9] as in human
cases, to escape many uncontrollable confounding factors. Rats were subjected to an IA model and the
incidence of IA lesions at the anterior or the posterior communicating artery complex and the onset of
SAH due to rupture of IA lesions at each site were examined.
In male rats, 15 among 18 animals (83.3%) developed IAs at the anterior or the posterior
communicating artery and 3 of these animals had multiple lesions at both artery complex. SAH
occurred in 5 among 18 rats or IA lesions (27.8%) during the observation period of 16 weeks after the
induction (Figure 1a–c). In female rats without the bilateral ovariectomy, 9 among 17 animals (52.9%)
developed IAs at the anterior or the posterior communicating artery and one of these animals had
multiple lesions. SAH occurred in 1 among 17 rats (5.9%) or 10 lesions (10.0%) in total in this group
(Figure 1a–c). In female rats with bilateral ovariectomy, 9 among 14 animals (64.3%) developed IAs at
the anterior or the posterior communicating artery and 1 of these animals had multiple lesions. SAH
occurred in 8 among 14 rats (57.1%) or 10 lesions (80.0%) in total in this group (Figure 1a–c). Although
the difference in sex or the implementation of ovariectomy did not influence the development of
IAs, the combination of these two factors significantly facilitated the rupture of induced IAs in a rat
model. The incidence of SAH per induced IA lesions was thus the highest in female rats with bilateral
ovariectomy in spite of the significantly lower systolic blood pressure in female rats than in male rats
(Figure 1).
3.2. Exacerbation of the IA Pathology in Female Rats with the Bilateral Ovariectomy
To explore mechanisms underlying the effect of sex difference or the implementation of ovariectomy
in female animals on the onset of SAH, the histopathological examinations of induced IA lesions
were done. The size of induced IAs was significantly larger in female rats with bilateral ovariectomy
than that in other groups (Figure 2a,b). Most IA lesions induced in female animals with bilateral
ovariectomy ruptured, resulting in SAH (Figure 1). Intriguingly, the remaining unruptured lesions
induced were apparently bigger than the ones induced in female animals without the ovariectomy or in
male animals (Figure 2a). Immunohistochemical analyses revealed the accumulation of MPO-positive
neutrophils and CD68-positive macrophages in arterial walls of the lesions, even in unruptured
IA lesions specifically from female animals with bilateral ovariectomy, similarly to ruptured ones
(Figure 2c). Such an accumulation of inflammatory cells was only limited in unruptured IA lesions
from male animals or female animals without ovariectomy (Figure 2c). Consistently, expression
of pro-inflammatory factor TNF-α, which is related with the pathogenesis [12–14], was higher in
unruptured lesions from female animals with bilateral ovariectomy than that in male animals or female
animals without ovariectomy (Figure 2d). In addition, expression of TNF-α in unruptured lesions
from female animals with bilateral ovariectomy was similar to that in ruptured lesions (Figure 2d).
In addition, the presence of vasa vasorum with SMA-positive media, which we identified as a
histopathological characteristic of ruptured IA lesions [9], could be detected even in unruptured
lesions only from female animals with the bilateral ovariectomy, and not in female animals without
ovariectomy or male animals (Figure 2e). The unruptured IA lesion induced in female animals withBrain Sci. 2020, 10, 335 5 of 11
the bilateral ovariectomy thus resembles ruptured lesions. In other words, the bilateral ovariectomy in
female
Brain Sci.animals
2020, 10, promotes
x FOR PEERevents
REVIEWunderlying rupture of the lesions. 5 of 11
Figure 1.1. The
Figure The incidence
incidence of of intracranial
intracranial aneurysms,
aneurysms, the the cumulative
cumulative incidence
incidence of of subarachnoid
subarachnoid
hemorrhage
hemorrhageand andthethe
rate rate
of rupture of induced
of rupture lesions. To
of induced induce To
lesions. intracranial
induce aneurysms,
intracranial10-week-old
aneurysms,
male (n = 18) or female Sprague−Dawley rats (n = 31) were subjected to the
10-week-old male (n = 18) or female Sprague−Dawley rats (n = 31) were subjected to the ligation ligation of the left carotidof
artery, thecarotid
the left right external
artery, carotid
the right artery and the
external right pterygopalatine
carotid artery and the right artery,pterygopalatine
and systemic hypertension
artery, and
by the combination
systemic hypertensionof aby high
the salt diet and the
combination of aligation
high saltofdiet
theand
left the
renal artery.ofInthe
ligation some female
left renal rats
artery.
(nIn=some
14), the bilateral
female rats (novariectomy was also
= 14), the bilateral applied. was
ovariectomy Animals were maintained
also applied. Animals were for 120 days after
maintained for
surgical manipulations. The incidence of intracranial aneurysms (a), the
120 days after surgical manipulations. The incidence of intracranial aneurysms (a), the cumulative cumulative incidence of
subarachnoid hemorrhage (SAH) (b), the rate of rupture of induced lesions
incidence of subarachnoid hemorrhage (SAH) (b), the rate of rupture of induced lesions (c) or (c) or systolic blood pressure
(sBP) (d) blood
systolic in each group; male
pressure (sBP) rats,
(d) infemale rats without
each group; ovariectomy
male rats, female rats(female)
without or ovariectomy
female rats with the
(female)
bilateral
or female ovariectomy
rats with the (ovariectomized), are shown.
bilateral ovariectomy Bars in (d) indicate
(ovariectomized), are shown. Bars ±
the mean inSEM. Statistical
(d) indicate the
analysis was done by the Tukey-Kramer method in (c,d). * p < 0.05, ** p < 0.01, ***
mean ± SEM. Statistical analysis was done by the Tukey-Kramer method in (c,d). * p < 0.05, ** p < 0.01, p < 0.001.
*** p < 0.001.
3.3. Disturbance in Endothelial Function by the Bilateral Ovariectomy in Female Rats
3.2. Exacerbation of the IAabout
A series of studies Pathology in Female
IAs has Ratsthe
clarified with the Bilateral Ovariectomy
involvement of macrophage-mediated chronic
inflammatory responses in the pathogenesis of the disease and also the potential contribution of
To explore mechanisms underlying the effect of sex difference or the implementation of
wall shear stress to this process [15–20]. To explore mechanisms regulating ovariectomy-mediated
ovariectomy in female animals on the onset of SAH, the histopathological examinations of induced
facilitation of rupture of lesions, we examined the effect of the bilateral ovariectomy on endothelial
IA lesions were done. The size of induced IAs was significantly larger in female rats with bilateral
cell function by using the stenosis model of the carotid artery in which the post-stenotic dilatation
ovariectomy than that in other groups (Figure 2a,b). Most IA lesions induced in female animals with
occurs in response to increased wall shear stress-loading [10,11]. In female rats without ovariectomy,
bilateral ovariectomy ruptured, resulting in SAH (Figure 1). Intriguingly, the remaining unruptured
the post-stenotic dilatation of the carotid artery could be observed at 30 min after the partial ligation,
lesions induced were apparently bigger than the ones induced in female animals without the
as expected (Figure 3a). However, in female animals with the bilateral ovariectomy, the post-stenotic
ovariectomy or in male animals (Figure 2a). Immunohistochemical analyses revealed the
dilatation was partially but significantly restricted (Figure 3a). Importantly, hormone replacement
accumulation of MPO-positive neutrophils and CD68-positive macrophages in arterial walls of the
therapy by Estradiol valerate restored ovariectomy-induced restriction of post-stenotic dilatation
lesions, even in unruptured IA lesions specifically from female animals with bilateral ovariectomy,
(Figure 3b). The bilateral ovariectomy thus disturbed endothelial cell function.
similarly to ruptured ones (Figure 2c). Such an accumulation of inflammatory cells was only limited
in unruptured IA lesions from male animals or female animals without ovariectomy (Figure 2c).
Consistently, expression of pro-inflammatory factor TNF-α, which is related with the pathogenesis
[12–14], was higher in unruptured lesions from female animals with bilateral ovariectomy than that
in male animals or female animals without ovariectomy (Figure 2d). In addition, expression of
TNF-α in unruptured lesions from female animals with bilateral ovariectomy was similar to that in
ruptured lesions (Figure 2d). In addition, the presence of vasa vasorum with SMA-positive media,which we identified as a histopathological characteristic of ruptured IA lesions [9], could be detected
even in unruptured lesions only from female animals with the bilateral ovariectomy, and not in
female animals without ovariectomy or male animals (Figure 2e). The unruptured IA lesion induced
in female
Brain Sci. 2020,animals
10, 335 with the bilateral ovariectomy thus resembles ruptured lesions. In other words,
6 of 11
the bilateral ovariectomy in female animals promotes events underlying rupture of the lesions.
Figure2.2.The
Figure Theexacerbation
exacerbationofofthe thepathology
pathologyofofintracranial
intracranialaneurysm
aneurysmininfemale
femalerats
ratswith
withthethebilateral
bilateral
ovariectomy.Ten-week-old
ovariectomy. Ten-week-oldfemale femaleSprague−Dawley
Sprague−Dawleyrats ratswere
weresubjected
subjectedtotothe
theaneurysm
aneurysmmodel.model.At At
120days
120 daysafter
afterthe
thesurgical
surgicalmanipulations,
manipulations,specimens
specimensofofinduced
inducedlesions
lesionswere
wereharvested.
harvested.(a,b)(a,b)The
The
macroscopic
macroscopicimageimage ofofinduced
induced lesions (a) (a)
lesions andandtheir sizesize
their (b) in
(b)each group;
in each malemale
group; rats (n = 18),
rats (n =female rats
18), female
without ovariectomy (female, n = 10) or female rats with the bilateral
rats without ovariectomy (female, n = 10) or female rats with the bilateral ovariectomy ovariectomy (ovariectomized,
= 10). Bar, 1.0 mm
n(ovariectomized, n =(a).
10). The
Bar,white
1.0 mm arrows and white
(a). The the black
arrowsonesand in (a)
theindicate
black onesthe in
unruptured
(a) indicate and
the
the ruptured lesions,
unruptured and therespectively.
ruptured lesions, Bars in (b) indicate Bars
respectively. in (b)±indicate
the mean SEM. Statistical
the mean analysis
± SEM.was done
Statistical
by the Tukey–Kramer
analysis was done by method. ***; p < 0.001.
the Tukey–Kramer (c,d) ***;
method. showpoccurs in response to increased wall shear stress-loading [10,11]. In female rats without ovariectomy,
the post-stenotic dilatation of the carotid artery could be observed at 30 min after the partial ligation,
as expected (Figure 3a). However, in female animals with the bilateral ovariectomy, the post-stenotic
dilatation was partially but significantly restricted (Figure 3a). Importantly, hormone replacement
therapy
Brain by 10,
Sci. 2020, Estradiol
335 valerate restored ovariectomy-induced restriction of post-stenotic dilatation
7 of 11
(Figure 3b). The bilateral ovariectomy thus disturbed endothelial cell function.
Figure 3. The Thedisturbance
disturbance of of
post-stenotic
post-stenoticdilatation by the
dilatation by thebilateral ovariectomy
bilateral ovariectomy in female rats.
in female
10-week-old
rats. female
10-week-old Sprague−Dawley
female Sprague−Dawley rats rats
werewere
subjected
subjected to to
thethebilateral
bilateralovariectomy
ovariectomy or or a
sham-operation. On
sham-operation. Onthethe7th7th
day,day, animals
animals underwent
underwent the carotid
the carotid ligationligation and the post-stenotic
and the post-stenotic dilatation
dilatation
was was for
observed observed for following
following 30 min (a). 30Inmin.
someIn some animals,
animals, hormone hormone replacement
replacement therapy
therapy (HRT) (HRT)
was
was applied
applied after after the bilateral
the bilateral ovariectomy
ovariectomy (b). Representative
(b). Representative macroscopic
macroscopic images images
of the of the carotid
carotid artery
artery (pre)
before beforeand
(pre) and after
30 min 30 mintheafter the (30
ligation ligation (30 shown.
min) are min) areThe shown. Theof
diameter diameter
the carotidof the carotid
artery was
artery wasbefore
calculated calculated
(pre), before (pre),
just after justand
(0 min) after
30(0
minmin)
afterand
the30 min after
ligation the ligation
(30 min). (30 min).
Bars indicate Bars
the mean
± SEM (nthe
indicate = 4). Statistical
mean ± SEM analysis was doneanalysis
(n = 4). Statistical by a Welch’s t test.by***;
was done p < 0.001.
a Welch’s t test. ***; p < 0.001.
3.4. Suppressive Effect of Sex Hormone on Inflammatory Responses in Macrophages and Neutrophils
Further, we examined the effect of the sex hormone, E2 (the hormone from the ovary) or DHT
(the hormone from the testis), on inflammatory responses by cultured macrophages (RAW264.7
cell line) or neutrophils (HL-60 cell line). In RAW264.7 cells, although LPS-induced expressions of
pro-inflammatory genes are related with pathogenesis, Tnf (TNF-α) [12–14], Il1b (IL-1β) [21], Ptgs2
(COX-2) [22] or Ccl2 (MCP-1) [20,23]—even with the pre-treatment by E2, the addition of E2 could
significantly suppress expressions of all of these genes compared with those in the vehicle-treated
cells (Figure 4a). The pre-treatment with DHT significantly suppressed expression of Ccl2 among four
genes examined (Figure 4a). In HL-60 cells, the pre-treatment of E2 or DHT suppressed LPS-induced
expression of pro-inflammatory genes (Figure 4b) in RAW264.7 cells. The suppressive effect of E2 was
stronger than that of DHT as well (Figure 4b). Consistently, expression of TNF-α was higher in lesions(COX-2) [22] or Ccl2 (MCP-1) [20,23]—even with the pre-treatment by E2, the addition of E2 could
significantly suppress expressions of all of these genes compared with those in the vehicle-treated
cells (Figure 4a). The pre-treatment with DHT significantly suppressed expression of Ccl2 among
four genes examined (Figure 4a). In HL-60 cells, the pre-treatment of E2 or DHT suppressed
LPS-induced
Brain Sci. 2020, 10,expression
335 of pro-inflammatory genes (Figure 4b) in RAW264.7 cells. The suppressive 8 of 11
effect of E2 was stronger than that of DHT as well (Figure 4b). Consistently, expression of TNF-α
was higher in lesions from female animals with bilateral ovariectomy than that in male animals or
from
femalefemale animals
animals with bilateral
without ovariectomy
ovariectomy (Figure than that in
2d). The male animals
results or vitro
of the in female animals
study without
suggest the
ovariectomy (Figure 2d). The results of the in vitro study suggest the suppressive
suppressive effect of the sex hormone on the inflammatory responses in lesions promotes effect of the sex
the
hormone
pathology. on the inflammatory responses in lesions promotes the pathology.
Figure4.4.Suppressive
Figure Suppressive effect
effect of sex
of the the hormone
sex hormone on expressions
on expressions of pro-inflammatory
of pro-inflammatory genes in genes
culturedin
cultured macrophages or neutrophils. RAW264.7 cells or HL-60 cells were
macrophages or neutrophils. RAW264.7 cells (a) or HL-60 cells (b) were pre-treated with β-estradiol pre-treated with
β-estradiol
(E2, 50 µg/mL) (E2,
or50 µ g/mL) or 5α-dihydrotestosterone
5α-dihydrotestosterone (DHT,
(DHT, 50 µg/mL) 50 hµ or
for 24 g/mL) for 24 h or 3 Cells
3 h, respectively. h, respectively.
were then
Cells were then stimulated with vehicle (Veh), LPS (dose) or TNF-α (dose) for
stimulated with vehicle (Veh), LPS (dose) or TNF-α (dose) for an additional 60 min. Expressions an additional 60 min.
of
Expressions of pro-inflammatory genes were examined by quantitative
pro-inflammatory genes were examined by quantitative RT-PCR analyses. Bars indicate the mean RT-PCR analyses. Bars±
indicate
SEM (n =the
4). mean ± SEM
Statistical (n = 4).was
analysis Statistical
done byanalysis was done by
a Tukey-Kramer a Tukey-Kramer
method. method.
*; p < 0.05, **; *; p***;
p < 0.01, < 0.05,
p<
**; pBrain Sci. 2020, 10, 335 9 of 11
by bilateral ovariectomy, which could be ameliorated by hormone replacement therapy [25,26]. In this
report, similar to the present study, the protective role of estrogen in endothelial cell function has been
indicated [26]. Furthermore, in human cases, the protective effect of hormone replacement therapy to
compensate for the defect in functions of the ovary on the onset of SAH was reported [4,27], suggesting
the clinical relevance of the present study. Hormone replacement therapy has adverse effects, such
as the increased risk of breast cancer, ischemic stroke and ischemic heart disease, which makes the
application of this therapy for the treatment of IAs in post-menopausal women controversial. However,
the present study has provided experimental evidence for the potential of hormone replacement
therapy as an option of treatment to prevent the onset of SAH in post-menopausal women.
Recent experimental studies mainly using an animal model of IAs [28,29] have clarified the
involvement of chronic inflammatory responses in the process regulating the initiation, progression
or rupture of IAs [15,16,18,30,31]. Additionally, hemodynamic force, especially wall shear stress,
is considered a mediator of IA formation and progression, mainly through a series of studies by
computational fluid dynamics analyses [17,32]. In the present study, we clarified the suppressive effect
of E2 or DHT on expressions of pro-inflammatory factors in cultured macrophages and neutrophils
(Figure 4). Additionally, the combination of female sex with bilateral ovariectomy exacerbated
inflammatory cells like macrophages or neutrophils in lesions (Figure 2). Here, the in vitro finding
that the suppressive effect of E2 was stronger than that of DHT may be responsible for the highest
incidence of rupture in female animals with bilateral ovariectomy. Furthermore, in the stenosis model,
the bilateral ovariectomy in female animals disturbed the high wall shear stress-induced post-stenotic
dilatation of the carotid artery (Figure 3), suggesting the malfunction of endothelial cells. Intriguingly,
the results of the present study have implied the role of the maladaptation of endothelial cells to shear
stress-loading at the bifurcation sites as a trigger of molecular events, leading to the progression and
rupture of the lesions. The ovariectomy in female animals, therefore, facilitates the pathogenesis of IAs
in multiple steps by influencing the functions of endothelial cells and inflammatory cells.
5. Conclusions
To explore mechanisms regulating rupture of IAs, we have used a rat model and revealed the
facilitation of the progression or rupture of the lesions by the combination of the female sex and the
bilateral ovariectomy. Furthermore, we have clarified the point of actions of sex hormone as endothelial
cells and inflammatory cells to inhibit the progression of the pathogenesis. The results of the present
study have thus provided new insights about mechanisms regulating the progression of the disease.
Author Contributions: Conceptualization, M.O. and T.A.; data curation, T.A.; formal analysis, M.O.; funding
acquisition, T.A.; investigation, M.O., I.O., K.S., M.K., H.M. and T.A.; methodology, M.O., I.O., K.S., M.K. and
H.M.; project administration, T.A.; resources, T.A.; software, M.O.; supervision, T.K.; validation, M.O., I.O., K.S.,
M.K. and H.M.; writing—original draft, M.O. and T.A.; writing—review and editing, T.A. All authors have read
and agreed to the published version of the manuscript.
Funding: This research was funded by Core Research for Evolutional Science and Technology (CREST) on
Mechanobiology from the Japan Agency for Medical Research and Development (AMED), grant number
JP18gm0810006 and JP19gm0810006.
Conflicts of Interest: M.K. was supported by CREST on Mechanobiology from AMED, grant number
JP18gm0810006 and JP19gm0810006, until 31 March, 2020. The other authors declare that they have no known
competing financial interests or personal relationships that could have appeared to influence the work reported in
this paper. The funders had no role in the design of the study; in the collection, analyses, or interpretation of data;
in the writing of the manuscript, or in the decision to publish the results.
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